Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the “scaffolding” polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated.P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c  and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha- and the glycerol-phosphorylated cyclic β--glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material.In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa.We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of  Pseudomonas aeruginosa is a Gram-negative bacterium, which can be found in soil, water, skin flora and many other natural and artificial environments. It is a cause of life-threatening lung infections in individuals with cystic fibrosis (CF), which are impossible to eradicate due to the biofilm lifestyle, adopted by this microorganism P. aeruginosa biofilms contains extracellular DNA P. aeruginosa biofilm matrix Biofilms are microbial communities that grow on a surface or at an interface, and are embedded in a thick extracellular (EC) matrix P. aeruginosaP. aeruginosa strains, representing most clinical and environmental isolates, do not produce much alginate but are still capable of forming fully mature biofilms P. aeruginosa genomic sequence revealed at least four additional gene clusters which appeared to encode proteins involved in exopolysaccharide biosynthesis. The structure of polysaccharide synthesis locus (psl)-dependent polysaccharide has been recently established pel (pellicle) gene cluster was shown to be involved in the formation of a pellicle, a surface-associated biofilm on the air-liquid (A–L) interface in standing cultures P. aeruginosa PA14 and its isogenic pel deletion mutant suggested that pel genes are responsible for the production if a glucose-rich matrix material P. aeruginosa PA14 and were coded by the ndvB, and not by the pel locus Alginate, an anionic polymer composed of mannuronic and guluronic acids, was traditionally considered the major EC matrix polysaccharide of mucoid P. aeruginosa PA14 is able to form thick, shear-forces resistant, biofilms at the A–L interface. These biofilms (or pellicles) are reported to have a rigid, paper-like consistency P. aeruginosa cell-wall. Indeed, the LPS structure of the common laboratory PA14 strain is still unknown. Subsequently, in an effort to identify the “scaffolding” polysaccharides component of P. aeruginosa PA14 biofilms, we performed a systematic analysis of the carbohydrate composition of the A–L biofilms of P. aeruginosa PA14. Knowledge of the nature of the EC polymers of PA14, which can potentially accumulate in the biofilm, could facilitate the task of establishing a correlation between putative genes encoding for key EPS biosynthesis and their products.The non-mucoid laboratory strain N-acetyl-quinovosamine, QuiNAc), and glucose (Glc); as well as small amounts of N-acetyl-glucosamine (GlcNAc) and heptose (Hep), characteristic for the lipid A-core region of LPS. Total fatty acid analysis showed the following main components: 2- and 3-hydroxydodecanoic acid ; n-hexadecanoic acid , and n-octadecanoic acid . Smaller amount of unsaturated octadecenoic acid  and fatty alcohols  were also present.We extracted LPS of PA14 cells by standard hot phenol-water method N-acetyl groups. This suggested that the polymer was composed of trisaccharide repeating units, with two sugar residues corresponding to 2-deoxy-2-aminosugars. 1H and 13C spectra of the O-PS were fully assigned using 2D homo- and heteronuclear correlation techniques. Spectra contained signals of a spin system of 2-acetamido-2-deoxy-α-L-galacturonic acid , substituted at position 4; and spin systems of 3-substitued 2-acetamido-2,6-dideoxy-α-D-glucose  and α-L-rhamnose . The sequence of the repeating unit (A–B–C) was established by inter-residue NOEs.LPS was hydrolyzed by acetic acid and the O-antigen polysaccharide (O-PS) purified on a Sephadex G-50 column. The detailed chemical structure of the O-PS was elucidated by NMR spectroscopy. The HSQC spectrum  indicateP. aeruginosa type O:2a,c Lanýi classification , 2-diacetamido-2,6-dideoxy-α- D-glucose ; and α-L-rhamnose   . TypicalP. aeruginosa cells often release large quantities of extracellular carbohydrates in the growth medium 1H-NMR and composition analysis -glucans We further carried out a systematic study of polysaccharides, produced by PA14 grown in standing cultures and in chemically defined M63 medium. Knowing that analysis  and 2. Tanalysis , and conCarbohydrate material of the growth medium was eluted as a large peak close to the void volume . It was P. aeruginosa PA14 in M63 medium.These results allowed us to conclude that LPS, the EC O-PS, and a family of cyclic glycerophosphorylated β--glucans are three main extracellular carbohydrates released by P. aeruginosa PA14 forms a rigid pellicle with a loosely adherent thick layer of a viscous transparent gelatinous EC matrix (GEC) (Growth in standing cultures in T-broth in Erlenmeyer flasks at room temperature are the conditions known as most favorable for the A-L biofilm formation of PA14 ix (GEC) . Upon ceix (GEC) . The GECKnowledge of the chemical structure of main carbohydrate polymers of PA14 enabled us to make the preliminary analysis of its A–L biofilm using isolation procedures which would minimize the loss of carbohydrate material .The soluble GEC detached by glass beads was dialysed, lyophilized and used for preliminary analysis . Cells aP. aeruginosa strains, in particular PAO1 The chemical composition of the GEC preparations was studied by colorimetric assays, DOC-PAGE, agarose gels, and monosaccharide composition analysis. Agarose gel profiles of the GEC preparations were identical to chromosomal DNA of this strain . The amoP. aeruginosa PA14. The detailed identification of this material requires further elucidation, and is currently under investigation.Furthermore, colorimetric assays and DOC-PAGE indicated that at least 20–25% of dry weight of this material consisted of LPS or LPS-like product. Interestingly, the DOC-PAGE profile of the GEC preparation was similar, but not identical, to the one of LPS extracted from cells by hot phenol-water method: In GEC, bands corresponding to the core-lipid A region were of relatively lower intensity, and O-antigen pattern indicated higher MW bands . Main mopelC mutant using the same procedures as for the wild-type (WT) strain. Monosaccharide composition, fatty acid and methylation analysis of corresponding fractions did not show significant differences between the two strains. Consistent with our previous studies pelC strain. eDNA was also detected in the GEC of the mutant strain  which favour an accumulation the EC carbohydrates, and in particular the Kdo-containing polymers, within the complex network of the A-L biofilm.We also attempted to estimate the correlation between the phenotypic difference of the WT and ΔC mutant . This maP. aeruginosa are the predominant environmental phenotype, and are also involved in colonization of CF lung at early stages of infection. It was shown that alginate is not a major component of the EC matrix of two nonmucoid laboratory strains, PAO1 and PA14 Nonmucoid strains of P. aeruginosa PA14 is a strain that has traditionally been used to study biofilms. This is largely due to its ability to form strong biofilm at the A–L interface. In the present work, we attempted to identify the carbohydrate polymers of the PA14 EC matrix. Knowledge of the nature of carbohydrate polymers which constitute the A–L biofilm of this strain could significantly facilitate establishing the identity of genes involved in their biosynthesis and, consequently, in the biofilm formation process.P. aeruginosa LPS, PA14 O-PS is an anionic polymer, rich in amino-sugars and containing 6-deoxy-sugar residues P. aeruginosa PA14, which is composed of trisaccharide repeating units with a structure identical to the LPS O-antigen.LPS is the major carbohydrate component of the cell envelope of Gram-negative bacteria, and has been proposed to be involved in bacterial attachment to abiotic surfaces and biofilm formation E. coli serotype O11 expressed a half of its O-antigen in the LPS-unlinked capsular form E. coli were assigned to Group 4 capsules Salmonella enteriditisFrancisella tularensisS. enteriditis of octadecanoic acid and unsaturated fatty acids, which are not normally seen in LPS of Gram negative bacteria and are rather common for phospholipids of bacterial membranes, led the authors to speculate that the lipid anchor for this CPS could be a phospholipid(s).Goldman and co-workers first showed that P. aeruginosa. However, a HMW immunogenic, non-toxic form of the LPS O-antigen has been previously isolated from culture supernatants of several P. aeruginosa strains Salmonella CPS P. aeruginosa PA14.To date, the presence of CPS has not been described for P. aeruginosa PAO1, similar gelatinous material of solid surface-associated (SSA) biofilms was reported to consist mainly of DNA P. aeruginosa biofilms.Knowledge of the chemical identity of the cell-wall LPS and EC carbohydrates allowed us to make a preliminary analysis of the composition of A–L biofilms of PA14. These biofilms formed in T-broth comprise rigid shear-forces resistant pellicle containing bacterial cells, and a loosely adherent transparent gelatinous material . In P. aAccumulation of LPS in the A-L biofilm is consistent with the physico-chemical properties expected for a molecular framework build up at the interface between air and liquid. Formation of an A-L biofilm would require the presence of components with both hydrophilic and hydrophobic properties. The amphiphilic LPS molecule with an anionic, viscous and highly hydrated O-PS, seems to be a good candidate for this function.P. aeruginosa biofilms P. aeruginosa LPS was shown to contribute to biofilm function and architecture by influencing bacterial adhesion, cell-to-cell adherence, and viscoelastic properties of biofilms et al.P. aeruginosa LPS is able to form stable monolayer at the air-water interface. It is therefore tempting to speculate that formation of such monolayer by the amphiphilic LPS molecules may occur in initial steps of pellicle formation. Otherwise, uppermost layers of the pellicle might be formed by cells with increased cell surface hydrophobicity. Interestingly, it is known that low concentrations of P. aeruginosa rhamnolipids cause a release of LPS from the outer membrane and an increase of the cell surface hydrophobicity algC, one of the alginate biosynthetic genes, whose expression is induced upon attachment to a surface P. aeruginosa.Previous studies pointed out LPS as a candidate molecule that could provide structural integrity of P. aeruginosaNeissseriaVibrio choleraeE. coli and Salmonella typhimuriumP. aeruginosa during normal growth occurs via the production of membrane vesicles The spontaneous release of LPS by Gram-negative bacteria during normal growth is a well-established phenomenon, which gave rise to the term “free endotoxin” Pseudomonas fluorescens and Salmonella spp. Salmonella is associated with the multicellular behaviour, which is characterized by the elaboration of thin aggregative fimbriae (Tafi), cellulose, and a yet uncharacterized EPS S. enteriditis was found to play an essential role in the protection of cells against desiccation stress via the formation of a hydrated gel, but did not affect the formation of the extracellular network between cells P. fluorescens, it was shown that cellulose fibers and LPS were required for strength and structural integrity of A-L biofilms, and defects in LPS expression affected the A-L biofilm strength P. aeruginosa. It is possible to hypothesise that cyclic β--glucans, which are known as good molecular chelating agents and are capable to bind to aminoglycoside antibiotic P. aeruginosa biofilms.To our knowledge, this and our previous study P. aeruginosa PA14: LPS-like material, EC O-PS and the cyclic β--glucan and provide the experimental evidence of accumulation of extracellular LPS-like material and cyclic β--glucans in the P. aeruginosa biofilm matrix. Understanding the involvement of these carbohydrates in the biofilm formation process of different non-mucoid P. aeruginosa strains is an attractive challenge and yet requires further investigation.In conclusion, in this work we identified three abundant extracellular carbohydrates of P. aeruginosa PA14 was used for all studies. Cells were grown in Erlenmeyer flasks  at 37°C for 3 days in M63 minimal medium; supplemented with 0.5% Casaminoacids ((Difco), 1 mM MgCl2, and 0.4% Glc −1 bacto peptone, 5 g l−1 NaCl). Standing cultures were inoculated with plate-grown bacteria to an OD600 = 0.0025, as recommended by Friedman and Kolter pelC mutant g, 4°C, 30 min). Joined aqueous and phenol phases were dialysed and lyophilised. The LPS preparation was further de-proteinated by TCA precipitation (5%), followed by dialysis and lyophilisation. This LPS preparation was used for a calibration curve in Kdo assays and as a control for DOC-PAGE.Cells were collected by centrifugation, washed with saline and extracted with 50% aqueous phenol at 65–70°C with intensive stirring For the preparation of the O-antigen, LPS was hydrolysed with 3% aqueous AcOH with stirring. Lipid A was removed by centrifugation, the supernatant lyophilised and fractionated on a Sephadex G-50 column. HMW fractions were collected and used for NMR analysis.2O and centrifuged ; clear supernatants were used for NMR analysis.The crude carbohydrate extract of the growth media and the extracellular biofilm matrix were prepared from M63 cultures and fractionated on Sephadex G-50 as described earlier g, 15 min). Clear supernatant was diluted with water, dialyzed and lyophilized. It was used for monosaccharide composition and methylation analysis, preceded by dephosphorylation . Alternatively to alkaline treatment, the pellicles were extracted with hot phenol-water as described above.For the chemical analysis of the pellicle, cell pellets which remained after mild sonication (rigid pellicle) were solubilized in 1 M NaOH at room temperature, with stirring for 10 min. The viscous solution was neutralized with glacial acetic acid, and the insoluble material removed by centrifugation . The remaining pellet was re-suspended in water and the procedure repeated. The clear supernatant represented the crude EC biofilm matrix solution and was used to assay the relative amount of LPS and DNA. Aliquots of this extract were used lyophilized to estimate the dry yield. The lyophilized material was further analyzed by agarose gel electrophoresis, DOC-PAGE, monosaccharide composition, fatty acid and methylation analysis. The remaining pellet (rigid pellicle) was solubilized in 1 M NaOH and analyzed as described above.Biofilm pellicles, formed in T-broth, were collected and washed gently with water. In order to detach the gelatinous extracellular material, they were suspended in water, and 5-mm glass beads were added to the suspension. The mixture was vortexed for 1 min, the cell suspension centrifuged .The eDNA from the growth media and GEC preparations was precipitated by 3 volumes of ethanol, collected by centrifugation and re-solubilized in water. Chromosomal DNA extractions were carried out on planktonic cultures of pelC mutant compared to the WT strain.3-ml cultures were transferred in the eppendorf tubes, and cell with cell-associated material was collected by centrifugation. Cell pellets were washed gently with saline (2 ml). Joined supernatants (“Growth media”) were dialyzed, lyophilized, and suspended in water (2 ml). Cell pellets were suspended in saline (2 ml) and subjected to sonication  to give a “sonication” extract. In a separate experiment, the pellet was solubilized in 1 M NaOH . In the three preparations, the amount of Kdo-containing material was assessed by the Kdo assay Gel-permeation chromatography was carried out on a Sephadex G-50 column , irrigated with water. Aliquots (200 µL) of each 5 mL fraction were assayed by colorimetry for aldose Methylation analysis and GC were performed as described before  1H and 13C NMR spectra were recorded using a Varian Inova 500 MHz and 400 MHz spectrometers as described before GC-MS was performed with a Hewlett Packard mass spectrometer 5989A equipped with a fused silica capillary column, as described before Relative amount of LPS in crude samples was estimated using the Kdo assay Figure S1P. aeruginosa PA14, grown in standing culture in M63 medium. Fractions obtained from cells, GEC and growth medium are color coded as explained in the legend of Schematic representation of the protocol used for purification of EC carbohydrates of (2.36 MB EPS)Click here for additional data file.Figure S2P. aeruginosa PA14, grown in standing culture in T-medium. T-medium is rich in complex carbohydrates and therefore was not analyzed. Fractions are color coded as explained in the legend of Schematic representation of the experimental protocol used for preliminary analysis of A-L biofilms of (1.87 MB EPS)Click here for additional data file.Figure S3P. aeruginosa PA14 A-L biofilm and purified chromosomal DNA. Lane 1, Ladder 100 pb; Lane 2, purified chromosomal DNA of P. aeruginosa PA14 WT, lane 3, purified chromosomal DNA of P. aeruginosa PA14ΔpelC; lane 4, eDNA of crude GEC extract of from P. aeruginosa PA14 WT; lane 5, eDNA of crude GEC extract of from P. aeruginosa PA14ΔpelC.Agarose gel-electrophoresis of eDNA of crude GEC extract of from (0.35 MB EPS)Click here for additional data file.


Because vascular endothelial growth factor-A (VEGF-A) is a key mediator in neovascularization response, we investigated the levels of the major pro-angiogenic  of each eye was measured via Tonopen in conscious rats. Eyes were enucleated either on the 5th or the 10th day of elevated IOP. Whole retinal lysates were separated by SDS–PAGE and transferred to PVDF membranes. Levels of VEGF-A164 levels remained unchanged between the control and glaucomatous retinas after five days (p=0.341) and 10 days of elevated IOP (p=0.117). The presence of the anti-angiogenic VEGF-A isoform has not been previously reported in the rat. An antibody specific to VEGF-A165b detected the anti-angiogenic protein in the rat retina. VEGF-A165b levels were significantly increased  in the glaucomatous retinas compared to those in controls after five days of elevated IOP. VEGF-A165b levels were not different (p=0.864) between the control and glaucomatous retinas following 10 days of elevated IOP. Expression of both VEGF-A164 and VEGF-A165b were observed in the retinal ganglion cells (RGC) and inner nuclear layer (INL).VEGF-A165b levels but not in the pro-angiogenic VEGF-A164 levels in the glaucomatous retina. VEGF-A165b levels return to baseline after 10 days of elevated IOP, and VEGF-A164 levels remain unchanged. We speculate that the short-term elevation of VEGF-A165b levels and/or the unchanged levels of VEGF-A164 contribute to the lack of neovascularization in the glaucomatous retina.Five day elevation of IOP leads to an increase in the anti-angiogenic VEGF-A Glaucoma is a neurodegenerative disease of retinal ganglion cells (RGC) that leads to blindness. Although the most prominent risk factor for RGC death in glaucoma is elevated intraocular pressure (IOP), the sequence of events by which IOP causes RGC death still remains largely unknown. One possible mechanism is that elevated IOP can induce abnormalities in blood flow in the glaucomatous eye. In open-angle glaucoma patients, abnormal vascular autoregulation has been observed in the inferior temporal retinal artery, the central retinal artery, the circulation of the optic nerve head, the choroid, and the perifoveal macular capillaries -8. It ha165 is the most abundantly expressed pro-angiogenic isoform in the retina [165b, an anti-angiogenic human VEGF-A isoform, has been shown to inhibit VEGF-A induced neovascularization in the mouse retina following ischemia [In the classical view of hypoxia, the ischemic tissue compensates for a decrease in oxygen levels by forming new blood vessels, a process known as neovascularization . VEGF-A e retina . More ree retina -20. For ischemia .There are only a few studies that have examined VEGF-A in glaucoma. VEGF levels were shown to be increased in the plasma of glaucoma patients when compared to that of healthy controls  and in t164 (the rat version of VEGF-A165) and anti-angiogenic VEGF-A165b (the rat version of VEGF-A165b) in normal and glaucomatous retinas after a short-term (five day) and an intermediate-term (10 day) elevation of IOP. Because of the lack of neovascularization in glaucoma, we hypothesized that the levels of VEGF-A165b but not VEGF-A164 would be increased in the glaucomatous retina.If ischemia contributes to the pathogenesis of glaucoma, why is there no neovascularization in glaucoma? To answer this apparent paradox, we investigated the levels of pro-angiogenic VEGF-AMale rats  were used for the study. Rats had ad libitum access to food and water during the study and were kept on a 12 h illumination cycle. All animal related procedures were performed in accordance with the statement for the use of animals in research released by the Association for Research in Vision and Ophthalmology.Rats (n=4) were anesthetized with an intraperitoneal injection of 1.5 mg/kg of acepromazine maleate, 7.5 mg/kg of xylazine, and 75 mg/kg of ketamine . Following shaving of the head, each rat was placed in a stereotaxic instrument. The skin covering the skull was incised along the midline using a surgical blade, and the skull was exposed and leveled. Next, for each hemisphere, a 30-gauge stainless steel needle was lowered into the superior colliculus at 5.3 mm posterior to the bregma, 1.5 mm lateral to the midline, and 4.8 mm ventral to the skull surface. Using a 5 μl syringe , 2 μl of Fluorogold solution  was injected over 10 min into each hemisphere. Following the injections, the skin was sutured. Rats were allowed to recover for a week before glaucoma was induced experimentally.To elevate IOP, hypertonic saline solution (1.9 M) was unilaterally injected in the episcleral veins as described by Morrison and colleagues . The conIOPs were measured with a TonoPen XL tonometer  in conscious rats . Measure2 inhalation either after five days or 10 days of elevated IOP. For western blotting, retinas were isolated from eyes obtained after five days and 10 days of elevated IOP (six pairs each). Retinas were placed in 200 μl of 1 mM of EDTA/EGTA/DTT, 10 mM of Hepes (pH=7.6), 0.5% Igepal , 42 mM of KCl, 5 mM of MgCl2, 1 mM of PMSF, and a tablet of protease inhibitors . After retinas were sonicated and incubated for 15 min on ice, samples were spun at 21,000 rpm at 4 °C for 30 min. Retinal proteins were quantified by spectrophotometry using the Bio-Rad Dc Protein Assay .Rats were sacrificed by COFor immunohistochemistry, four pairs of eyes that were enucleated after five days of elevated IOP were fixed with 4% paraformaldehyde for 20 min at room temperature. These eyes were previously back-labeled with Fluorogold. Next, the posterior eye cups were isolated and fixed with 4% paraformaldehyde for an additional 40 min at room temperature. After posterior eye cups were cryoprotected overnight in graded sucrose dilutions, they were placed in the optimal cutting temperature compound  and were sectioned 16 μm thick using a cryostat.164 protein  was also loaded as a positive control in certain experiments. Proteins separated by SDS–PAGE were then transferred to polyvinylidene difluoride membranes  for 1 h. After the membrane was blocked for 1 h at room temperature with 2% ECL Advance Blocking Agent  in Tris-buffered saline with Tween (TBS-T), it was incubated at 4 °C overnight with a primary antibody. The primary antibodies used in this study were as follows: rabbit polyclonal anti-VEGF , mouse monoclonal anti-VEGF165B , and mouse monoclonal anti-α-tubulin . The next day, membranes were incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies. Goat anti-rabbit IgG  and goat anti-mouse IgG  were the secondary antibodies used in the study. Both of these antibodies had minimal cross-reaction to rat serum proteins. Next, membranes were processed with ECL Advance Western Blotting Detection Kit  and exposed to Kodak BioMax Light Film .Retinal proteins isolated after either five days or 10 days of elevated IOP were separated on Tris-HCl Ready-Gels . Recombinant rat VEGF-At-test was used to evaluate the significance of the ratios for a given protein . Data were reported as mean±standard error of the mean (SEM) in the text.A Personal Densitometer SI  was used to scan the exposed films. The density of the protein of interest on the film was measured using ImageQuant 1.2 (Molecular Dynamics). First, the background density was subtracted from the density of each band. Next, the densitometric reading of the protein of interest was normalized to α-tubulin readings, which served as loading controls. For each retina pair, the normalized densitometric reading from the glaucomatous retina was divided by the reading from the control retina. Then, the ratios from different pairs of retinas were averaged. For statistical analysis, a one-sample Retinal sections from four pairs of eyes were blocked for 1 h at room temperature in 4% normal goat serum and 0.3% Triton-X 100 in 1X PBS, pH 7.4. Sections were incubated with the primary antibody overnight at 4 °C. Some sections were incubated in blocking solution without the primary antibody and were used as negative controls. The primary antibodies and the dilutions used in the study were mouse monoclonal anti-VEGF  and mouse monoclonal anti-VEGF165B . The following day, sections were incubated with the Alexa Fluor 594-conjugated goat anti-mouse secondary antibody  for 1 h at room temperature. After the sections were treated with Prolong Gold anti-fade reagent, staining was visualized using an Olympus BX51 microscope .On our BX51 microscope, we used UPlanApo 0.70 NA 20X (Olympus) and UPlanApo 0.85 NA 40X (Olympus) objective lenses through a 10X ocular or camera lens to image our retinal sections for a total magnification of 200X or 400X, respectively. Excitation/emission filter cubes used for a given fluorophore were 11006v2 Gold  for Fluorogold and N41004 HQ Texas Red (Chroma) for Alexa Fluor 594. We used DPController 1.2.1.108 (Olympus) in conjunction with the DP70 color camera (Olympus) affixed to the BX51 to image the retinal sections for fluorescence. We then used Adobe Photoshop to layer the images and apply transparency to see the overlap of different fluorophores in a given section area.Average peak IOP was (mean±SEM) 40.7±1.9 mmHg and 43.1±0.8 mmHg for the five-day (n=6) and 10-day (n=6) groups that were used in the western blot (WB) analysis, respectively. Average peak IOP was 43.4±0.8 mmHg for the other five-day group used for the immunohistochemistry (IHC) analysis (n=4).164 dimer in all retinas and in the positive control brain  of the recombinant rat VEGF-A164 protein with the anti-VEGF-A165b antibody. Whereas the anti-VEGF-A165b antibody did not recognize the VEGF-A164 protein at any concentration, it recognized two bands around 22.4 and 45 kDa in a pair of control and glaucomatous retinas corresponding to the monomer and dimer forms of VEGF-A165b, respectively , VEGF-A165b monomer levels were significantly increased in the glaucomatous retinas compared to those in controls   or for the VEGF-A165b monomer  between the control and glaucomatous retinas  . However retinas . These r165b was similar to that of VEGF-A164. VEGF-A165b staining was observed in the RGC and in the inner nuclear layer (165b immunoreactivity in the glaucomatous retinas compared to normal retinas (IHC demonstrated that the distribution of VEGF-Aar layer . In the ar layer . No staiar layer . Consist165b is present in the rat. In particular, we show that VEGF-A165b is present in the retina and localized primarily to the RGC layer and the inner nuclear layer. Our findings for VEGF-A165b show a distribution similar to that seen for VEGF-A164 in this report and previous reports about VEGF-A164 by others [165b. Our data show that VEGF-A165b levels are increased early in the cause of experimental glaucoma but return to baseline at a later time point. IHC results show that this increase is primarily due to increased expression in the RGC layer and in the INL.We demonstrate in this report that VEGF-Ay others ,29. Usin164 do not change in the glaucomatous retina compared to control retinas in the rat after five days or 10 days of elevated IOP. Consistent with previous studies, we observe that VEGF-A164 is expressed in the RGC and INL of the retina [Our results demonstrate that the levels for the pro-angiogenic VEGF-Ae retina ,29.165 is the most abundantly expressed pro-angiogenic isoform in the retina [165 and VEGF-A165b mRNA are produced from the VEGF-A pre-mRNA via alternative splicing [165 and VEGF-A165b share a 96.4% homology and differ only in the last six amino acids in their amino acid sequence in humans [165 is pro-angiogenic, VEGF-A165b has an inhibitory effect on angiogenesis both in vitro and in vivo [165b inhibits neovascularization in the mouse retina following oxygen-induced retinopathy [165b is observed to be the dominant isoform [VEGF-Ae retina . Both VEsplicing ,18. VEGF in vivo . For exainopathy . More reinopathy ,20. It h isoform ,18.165b mRNA in the glaucomatous retina? Although the precise answer remains unknown, proposed mechanisms include differential promoter selection, alternate regulation of mRNA stability, and regulation of alternative splicing [VEGF-A gene is being transcribed, the emerging pre-mRNA is instantaneously processed by several RNA-binding proteins and splice factors [What molecular mechanism is responsible for the upregulation of VEGF-Asplicing ,30-33. I factors . These p factors . It is t factors ,35,36. M factors . For exa factors ,39. In a factors .165b but not the pro-angiogenic VEGF-A164 in our experimental glaucoma model. The combination of these findings suggests that the elevation of VEGF-A165b levels and/or the unchanged levels of VEGF-A164 contribute to the lack of neovascularization in the retina in glaucoma.In conclusion, we report an increase in the retinal levels of the anti-angiogenic VEGF-A


TNF-α gene variation was associated with risks of subarachnoid hemorrhage in multiple ethnicities. The present case-control study tested the hypothesis that genetic variations of the TNF-α gene may affect the risk of Taiwanese SDICH. We examined the association of SDICH risks with four single nucleotide polymorphisms (SNPs) within the TNF-α gene promoter, namely T-1031C, C-863A, C-857T, and G-308A.Genetic factors may play a role in susceptibility to spontaneous deep intracerebral hemorrhage (SDICH). Previous studies have shown that Genotyping was determined by PCR-based restriction and electrophoresis assay for 260 SDICH patients and 368 controls. Associations were tested by logistic regression or general linear models with adjusting for multiple covariables in each gender group, and then in combined. Multiplicative terms of gender and each of the four SNPs were applied to detect the interaction effects on SDICH risks. To account for the multiple testing, permutation testing of 1,000 replicates was performed for empirical estimates.In an additive model, SDICH risks were positively associated with the minor alleles -1031C and -308A in men  but inversely associated with -863A in females . There were significant interaction effects between gender and SNP on SDICH risks regarding SNPs T-1031C, C-863A, and G-308A . Hemorrhage size was inversely associated with -857T in males (p = 0.04).TNF-α gene promoter with SDICH risks are gender-dependent.In the Taiwan population, the associations of genetic variations in the  Deep parenchymal structure including the basal ganglia, thalamus, brainstem, and cerebellum is the most frequently 65% to 80%) affected site of spontaneous intracerebral hemorrhage (SICH) ,2. SICH % to 80% ACE, APOE, neprilysin, endoglin, TGF-β1), endothelial dysfunction (ACE), inflammation markers , and hemostasis . However, these studies were mainly conducted in lobar SICH. In contrast, the number of genetic studies of SDICH is limited [A recent article comprehensively reviewed the candidate genes of SICH reported during 1996 to 2007 [ limited -10.As a complex disease, SDICH aggregate in families but does not segregate in a mendelian fashion ,6,11. Faα (TNF-α) is one of the main proinflammatory cytokines and plays a central role in initiating and regulating the inflammatory response. The TNF-α gene is located in 6p21.3. Previous studies have shown that polymorphisms at position -308 and -863 in the promoter region of the TNF-α gene were associated with risks of subarachnoid hemorrhage (SAH) in multiple ethnicities [α concentrations may contribute to this association [TNF-α polymorphism with SICH in a Japanese study [TNF-α polymorphism with SDICH risk.Concentrations of inflammation markers in plasma and cerebral spinal fluid have been shown to be positively correlated with mortality in SICH patients . Tumor nnicities ,16. Diffociation ,18. In cTNF-α gene would predispose to SDICH and modify the 30-day outcome of SDICH events in the Taiwan population.The purpose of this study is to evaluate whether any particular polymorphism of This is a retrospective case-control study. Subjects were recruited from the Department of Neurology, Chang Gung Memorial Hospital (CGMH), Linkou Medical Center, which is a tertiary referral center serving residents mainly in the northern Taiwan. Patients with SDICH were diagnosed based on both clinical presentations and brain computed tomography (CT) ,10,19. CAnthropometric data and 12-hour fasting blood were collected from all participants. For SDICH cases, blood was drawn within one week of event in average. Diabetes mellitus (DM) was defined based on World Health Organization (WHO) criteria. Hypertension was diagnosed when blood pressure (BP) repeatedly exceeded 140 mm Hg (systolic) and/or 90 mm Hg (diastolic) or when a subject was taking antihypertensive medication to control hypertension. For SDICH cases without prior diagnosis of hypertension, patients were considered to have hypertension when BP measured after the acute phase of SDICH (14 days within the onset of hemorrhage) repeatedly exceeded 140 mm Hg (systolic) and/or 90 mm Hg (diastolic). Body mass index (BMI) was calculated by weight in kilograms divided by squared height in meters. Alcohol use was defined as drinking ≥210 g per week.Hemorrhage located at the basal ganglia and thalamus was defined as supratentorium SDICH, while brainstem and cerebellum hemorrhage were defined as infratentorium SDICH. Volume of the hematoma was calculated using the so-called ABC method based on CT of brain . Short tTNF-α T-1031C, C-863A, C-857T, and G-308A were determined by PCR-based single-strand conformation polymorphism (SSCP) and/or restriction fragment length polymorphism (RFLP) assays. The details of specific PCR conditions and detection for genotyping, including polymorphism primer pair, anneal, and enzyme were described in our previous report [Genomic DNA was isolated from peripheral leukocytes using DNA Extraction Kit (Stratagene). Genotypes for s report . The amp2 test with significance level of 0.001 in each of the case and control groups and in the combined group. Patterns of linkage disequilibrium (LD) were evaluated using Haploview v4 [Each of the four single nucleotide polymorphisms (SNPs) was checked for Hardy-Weinberg equilibrium (HWE) using the standard χoview v4 , and hapoview v4  based on2-test or t-test was utilized to compare demographic data and the distributions of genotypes of TNF-α between controls and cases. Two-tailed P-values were derived from the χ2-test or Fisher's exact test. Association analyses were performed first stratified by genders, and then in combined. Multivariable logistic regression was used to analyze the phenotype-genotype associations of SDICH with TNF-α alleles under additive genetic models. Covariables included age, sex, BMI, and hypertension, total cholesterol level, and alcohol use [The Pearson's χohol use . BecauseIn the present case-control study, at the 5% significance level, we had power greater than 0.8 to identify an association under an additive genetic model when the per-allele genetic effect was greater than an odds ratio of 1.5 and the disease allele frequency was greater than 0.2.All the data analyses were performed using SAS software version 9.1.3 .The characteristics of the study groups are presented in Table 2 test, p = 0.01 and p = 0.02, respectively). In contrast, the distribution of C-857T differed significantly between cases and controls in females  but not in males. Each of the four SNPs was in HWE in each of the case and control groups and in the combined group. The minor allele frequencies of all the four SNPs were ≥ 0.05, and therefore we utilized the four SNPs to evaluate LD. There was a single block structure, composed of a portion of the SNPs T-1031C and C-863A with D' estimation of 0.92.In the entire cohort, distributions of all the four SNPs did not differ between SDICH cases and controls Table . When stWhen adjusting for age, sex, BMI, hypertension, TC level, and alcohol use, logistic regression showed associations of the four SNPs with SDICH risks in a different direction in each gender group. Specifically, in an additive model, SDICH risks were positively associated with the minor alleles of SNPs -1031C and -308A in men  but inversely associated with -863A in women . These associations were not significant in the combined cohort because of the opposite direction of associations in each gender group. Multiplicative terms of SNPs and gender evaluated in the same additive models showed significant SNP-gender interaction effects on SDICH risks regarding SNPs T-1031C, C-863A, and G-308A . Haplotypes constructed by the SNPs T-1031C and C-863A were not associated with the SDICH risks in each gender group and in the combined group (data not shown).2 test) (Table 3) than in females . There was no association of hemorrhage location with any of the four SNPs . Regarding hemorrhage size, only SNPs C-857T showed a borderline association in male group after adjusting age, BMI, alcohol use, and hypertension in additive model . No significant SNPs-gender effect on hemorrhage size was found. Haplotypes constructed by the SNPs T-1031C and C-863A were not associated with hemorrhage size in each gender and in the combined group.In the SDICH group, 149 in 177 (84.2%) males and 80 in 83 (96.4%) females had hemorrhage located at supratentorium  and hematoma size (p < 0.001) (data not shown in tables), but neither with any of the four SNPs nor the constructed haplotypes in the male, female, and in the combined groups. There was no SNP-gender interaction effect on short term outcome.a priori knowledge of gene function, modest genetic effect, polygenic effect, and epistasis. The study herein shows that the common genetic variations of TNF-α gene are associated with SDICH risks with significant gender differences. The direction of associations between TNF-α gene and SDICH is opposite between genders. In an additive model, SDICH risks are positively associated with the dose of minor alleles of -1031C and -308A in men, but inversely associated with -863A in female. Multiplicative terms of analyses confirm the interaction effects between gender and each of the SNPs T-1031C, C-863A, and G-308A on SDICH risks. Hemorrhage size is inversely associated with -857T allele in males. No association between the 30-day outcome and TNF-α gene is found in this study. Haplotypes constructed by the SNPs T-1031C and C-863A did not reveal any association with SDICH risks, hemorrhage size, and mRS.Knowledge of susceptibility genes of SDICH, a common complex disease, is largely lacking because of the study obstacles of the need of α, one of the main proinflammatory cytokines regulating an inflammatory response, has been demonstrated to be associated with increased mortality rate of SICH [Increased serum TNF- of SICH  and recu of SICH . Previou of SICH  and the  of SICH  are asso of SICH  consiste of SICH . This st of SICH ,16. Loba of SICH . The maj of SICH ,12, whil of SICH . In addiα concentration. The SNPs -308A [TNF-α expression. Possible mechanisms of the influence may come from substances binding to regulatory elements, such as nuclear factor κB and organic cation transporter 1, and alteration of the secondary structure of DNA to affect accession of cis-acting transcription factors to the promoter/enhancer region of the TNF-α gene [TNF-α and SDICH risks. In males, we found that minor alleles of the TNF-α variation are risks to SDICH as hypothesized. However, the direction of the association was opposite in females. Several clinical and experimental studies have shown gender differences in the cytokine response to stress [α are increased in men as compared to women after experimental lymphocyte stimulation in an ex vivo study [Polymorphisms in the regulatory region of promoter may result in different TNF-Ps -308A , -1031C,Ps -308A  have beeF-α gene ,27. The o stress ,29. Typevo study .α by stimulating the release of plasminogen activator inhibitor type 1 and by reducing the release of t-PA through the extrinsic route [α may be responsible for the early activation of hemostatic mechanism during a SDICH event [α, which may in turn decrease the hemorrhage size.Our study also showed an inverse association of -857T with hemorrhage size in males. Because of the antifibrinolytic effects of TNF-ic route , the relCH event . The -85α concentration, which may otherwise provide additional functional information to support our hypothesis. A further study examining the correlation between the SNPs and serum TNF-α concentration will be useful. Nevertheless, our study has several strengths. This study included a homogenous disease entity in a same ethnic background, which may limit the confounding effect from multiple phenotypes and ethnicities. This is the first study analyzing associations between TNF-α genotype and the SDICH susceptibility, and independent confirmation is needed to verify these associations.It is worth mentioning that patients in this study were ascertained from the Department of Neurology, which might cause a smaller average hemorrhage size than that of patients in the Department of Neurosurgery. Furthermore, this study does not have sufficient subjects of infratentorium hemorrhage to examine the correlation between genetic effects and the significant gender difference in the hemorrhage loci. Also, the number of female cases is relatively small, which is due to a lower incidence rate compared to males  rather tTNF-α polymorphisms on SDICH risks and size with significant gender differences.This study showed a modest effect of The authors declare that they have no competing interests.YCC contributed to study design, analysis, interpretation of data, and drafted the manuscript. FJH carried out the molecular genetic studies. PBC carried out the molecular genetic studies. YRW contributed to acquisition of data. HCW made substantial contributions to conception and design. STC participated in the design of the study and has been involved in drafting the manuscript. GJLC made substantial contributions to the molecular genetic studies. CMC contributed to study design, molecular genetic studies, interpretation of data, and has given final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/10/41/prepub


A series of studies showed the presence of substantial amount of nerve fibers and their close relationship with the anterior pituitary gland cells. Our previous studies have suggested that aside from the classical theory of humoral regulation, the rat anterior pituitary has direct neural regulation on adrenocorticotropic hormone release. In rat anterior pituitary, typical synapses are found on every type of the hormone-secreting cells, many on lactotrophs. The present study was aimed at investigating the physiological significance of this synaptic relationship on prolactin release.The anterior pituitary of rat was sliced and stimulated with electrical field in a self-designed perfusion chamber. The perfusate was continuously collected in aliquots and measured by radioimmunoassay for prolactin levels. After statistic analysis, differences of prolactin concentrations within and between groups were outlined.The results showed that stimulation at frequency of 2 Hz caused a quick enhancement of prolactin release, when stimulated at 10 Hz, prolactin release was found to be inhibited which came slower and lasted longer. The effect of nerve stimulation on prolactin release is diphasic and frequency dependent.The present in vitro study offers the first physiological evidence that stimulation of nerve fibers can affect prolactin release in rat anterior pituitary. Low frequency stimulation enhances prolactin release and high frequency mainly inhibits it. It has been well acknowledged that the anterior pituitary is regulated via hypothalamic hormones released at the median eminence . AlthougHaving observed synapses on corticotrophs -13, we sIn the rat, synapses are found on every hormone-secreting cell type. In fact, it is the lactotroph that is most frequently found to have a synapse . ProlactAdult male Sprague-Dawley rats, weighing 220-240 g, were obtained from FMMU's University Laboratory Animal Center. They were housed (three rats per cage) in a temperature-controlled room, and a 12: 12 h light/dark cycle (lights on at 07.00 h), with free access to food and water.The rats were handled by holding and gripping 10 times each day for 5 consecutive days before experiments to minimize the effect of stress. Every precaution was taken to ensure proper and humane treatment of the rats. The number of rats used in each experiment group is indicated in the figure legends and tables.2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.0, Glucose 11.0, plus BSA 0.1% and bacitracin 40 mg/L) and rinsed three times. The anterior pituitary was then cut sagittally into 0.8 mm slices with a self-designed tissue chopper. The slices were subsequently transferred into a self-designed stimulation-perfusion chamber.The experiments started at 09.00 h, the rats were anaesthetized with sodium pentobarbital . After decapitation, the pituitary gland was immediately dissected. The intermediate and posterior lobes were then removed and discarded. The completeness of the removal was checked by hematoxylin and eosin (H-E) staining. The isolated anterior lobe was kept in pre-cooled (4°C) Krebs-Ringer bicarbonate solution  and applied through two ring-shaped electrodes sandwiching the tissue slices. Based on our previous experience , square The double-antibody rat prolactin RIA kit  was used for the measurement of prolactin concentrations. The range of assay was 10-200 pg/ml, with the intra- and inter-assay variation being 5.6% and 8.9% respectively. Measurements in duplicates were performed.Dunnett t -test. Differences between groups were determined by the least significant difference test (LSD). P < 0.05 was considered statistically significant .For statistic analysis, SPSS statistical software, version 10.0  was used. Significance of differences was determined by analysis of variance (ANOVA). Differences in changes of prolactin concentration within groups were determined by the To test the basal release of prolactin during the experiment, the anterior pituitary slices were perfused with KRBGA only and the perfusate was continuously collected every 10 min or every 1 min.To test the effect of TTX alone on prolactin secretion, TTX  of 1 μmol/L was added to KRBGA after stabilization with KRBGA perfusing for 30 min. The perfusate was continuously collected every 10 min or every 1 min.To block the action potential along the nerve fibers, TTX of 1 μmol/L was added to KRBGA, and the anterior pituitary slices were pre-incubated for 30 min before EFS started. To compare with the experimental groups, the perfusate was also continuously collected every 10 min or every 1 min.+ and 64.9 mmol/L of Na+) at the end of each test, to evoke peak prolactin output. To detect the concentration of prolactin, these samples were diluted with 0.85% NaCl solution before RIA.For assessment of the functional viability of lactotrophs during the experiment, KRBGA was replaced with high potassium KRBGA . In 1-min samples , peaked at sample-3 and sample-4 , and then subsided until sample-7, when the prolactin secretion returned to the basal level.The EFS started at the beginning of sample-0. When analyzed in 10-min samples Fig. , there wP < 0.01 for all). At sample-30, it declined to 127% (P < 0.01) and then returned to the basal level at sample-40 , prolactin secretion level maintained as a plateau until the end of the 10-min experiment Fig. , the incP < 0.01).Statistic analysis proved that there was significant difference between 2-min and 10-min stimulation groups (P < 0.01), which was followed by an inhibition of 3 min, from sample-4 to sample-6 . The prolactin concentration resumed basal level from sample-7 on , the same as in 2-min stimulation experiment. Similarly, the inhibition started from sample-4, but it lasted till the end of the experiment (sample-9) with a very gentle down slope . The 10-min sample analysis , with the lowest value at samples-20 and 30.In 1-min samples Fig. , an earlP < 0.01).Statistic analysis showed that there was significant difference between 2-min and 10-min stimulation groups  along with a smaller quantity of classical transmitter ,21. We bThe present in vitro study offers the first physiological evidence that stimulation of nerve fibers can affect prolactin release in rat anterior pituitary. Low frequency stimulation enhances prolactin release and high frequency mainly inhibits it.KRBGA: Krebs-Ringer bicarbonate solution; EFS: electrical field stimulation; TTX: tetrodotoxin; RIA: radioimmunoassay; min: minute(s); PRL: prolactin; ACTH: adrenocorticotropic hormone.The authors declare that they have no competing interests.PZ carried out the main experiments and wrote the first draft of the manuscript. LL performed the RIA. CJX performed the statistical analysis. KHW structured and proofread the final manuscript. LZG and GJ designed the study and wrote the final manuscript. All authors read and approved the final manuscript.


Escherichia coli lipopolysaccharide (LPS) in the monocyte-derived macrophage THP-1 cells demonstrated that the URG-treated cells showed a significant reduction in cytokine expression than the RG-treated ones. Transcription expression of the LPS-induced cytokines such as TNF-α, IL-1β, IL-6, IL-8, IL-10, and TGF-β was significantly inhibited by URG compared to RG. These results suggest that some biologically active and soluble components in red ginseng can be more effectively extracted from URG than RG by standard hot-water extraction.Red ginseng is one of the most popular traditional medicines in Korea because its soluble hot-water extract is known to be very effective on enhancing immunity as well as inhibiting inflammation. Recently, we developed a new technique, called the HAC-gearshift system, which can pulverize red ginseng into the ultra-fine granules ranging from 0.2 to 7.0 μm in size. In this study, the soluble hot-water extract of those ultra-fine granules of red ginseng (URG) was investigated and compared to that of the normal-sized granules of red ginseng (RG). The high pressure liquid chromatographic analyses of the soluble hot-water extracts of both URG and RG revealed that URG had about 2-fold higher amounts of the ginsenosides, the biologically active components in red ginseng, than RG did. Using quantitative RT-PCR, cytokine profiling against the  Panax, Panax ginseng, so called “Korean ginseng”, is the most popular ginseng in Korea. Several studies on P. ginseng suggest that it has many beneficial effects such as anti-inflammation, anti-oxidation, and anti-cancer activity [P. ginseng is known to be the ginsenosides (saponins) triterpene glycosides. Many different kinds of ginsenosides have been reported, but their amounts and composition can be varied depending on the types of P. ginseng such as undried, dried (white), or red ginseng [Among the several species of the genus activity . A well  ginseng . Red ginBy definition, cytokines are a large group of soluble extracellular proteins or glycoproteins which are functioning as intercellular signals and/or effectors. Based on the structural homologies of their receptors, they are classified into three family groups, namely interleukins (IL), interferons (IFN), and chemokines. In function, however, cytokines can be divided into the two groups; pro- or anti-inflammatory cytokines. The pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-1α/β, IL-12, and IFN-γ induce immunogenic and inflammatory responses –5, whereRecently, a new technique has been developed to make medicinal herbs into very fine particles with sizes ranging from 0.2 to 7.0 μm. This simple, but innovative pulverization technique has been named the HAC-gearshift system because the system involves a total of three consecutive pulverization steps; the first is in a hammer mill, the second in an air classifying mill, and the third in a classifying cyclone. The average size of the final particles through this system ranged from 0.2 to 7.0 μm. In contrast, the conventional pulverization technique produces relatively larger particles than the HAC-gearshift system does, with sizes averaging 127 μm.In this study, we hypothesized that this physical modification from normal to ultra-fine granules of medicinal herbs by the HAC-gearshift system could enhance their medicinal effects. To test this hypothesis, the ultra-fine granules of red ginseng (URG) were formulated using the HAC-gearshift system and used to prepare the soluble extract by standard hot-water extraction. Finally, the soluble hot-water extract of URG was compared to that of the normal granules of red ginseng for their biochemical compositions and previously defined anti-inflammatory activities.Recently, we developed a new pulverization technique called the HAC-gearshift system, which allows producing very fine particles in sizes ranging from 0.2–7.0 μm . Using tTherefore, we hypothesized that URG might be more efficient than RG for extracting some beneficial components of red ginseng because of their particle size differences. To test this, the soluble extracts from either URG or RG were prepared by standard hot-water extraction  and measThe results showed that the hot-water extract of URG contained about 2-fold higher amounts of the ginsenosides than that of RG. As a total, about 20.9 mg/g of the ginsenosides was extracted from URG, but only about 10.7 mg/g was extracted from RG. This indicates that URG can be more efficiently extracted by hot-water extraction than RG .Previous studies reported that the hot-water extract of RG had a significant anti-inflammatory effect and its biologically active components were the ginsenosides. Since the hot-water extract of URG contained significantly higher amounts of the ginsenosides than that of RG, it is possible that the URG extract may have higher anti-inflammatory activity than the RG extract. Therefore, we determined the abilities of URG and RG to induce the pro-inflammatory cytokines. To the end, any potential toxic effects of URG and RG were examined at first by measuring cell viability over the monocyte-driven macrophage THP-1 cells , 12. UsiSince it is known that transcription expression of TNF-α is induced by LPS in the THP-1 cells unless the cells are differentiated by phobol 12-myristate 13-acetate (PMA), we examined transcription expression of TNF-α in the THP-1 cells with or without PMA, LPS, or both. Using quantitative RT-PCR, the results support the fact that TNF-α expression was not induced in the THP-1 cells without pre-treatment of PMA. The PMA-treated cells with LPS for 2 h could induce substantial expression of the TNF-α mRNA . In contIn order to examine the effects of URG and RG on LPS-induced cytokine expression, transcription expression of the previously defined pro-inflammatory cytokines was examined by quantitative RT-PCR. The THP-1 cells were exposed to PMA for 72 h and then treated with LPS with or without URG, RG, or both as described above. As expected, both URG and RG extracts were able to down regulate expression of both pro- and anti-inflammatory cytokines including IL-1β, IL-6, IL-8, TNF-α, IL-10, and TGF-β . The obsThe ultra-fine granule is a basic feature of our HAC-gearshift system, whereby food or medicinal herbs are physically formulated as nano-particles by a broken cell wall technology. In order to evaluate and compare this system to the conventional pulverization system, red ginseng was applied to this system and compared to that prepared by the conventional pulverization system. When URG was compared with RG, we found that the soluble hot-water extracts of URG had more ginsenosides, the biologically active components of red ginseng, than those of RG as well as showed enhanced inhibitory effect on LPS-induced cytokine expression in THP-1 cells compared to those of RG.in vitro. This inhibitor was discovered by screening a chemical library for inhibitors of the MAP kinase cascade [Previously, it was well known that LPS, the endotoxin component of Gram-negative bacteria, is responsible for initiating the host responses causing inflammation . This mo cascade . AlthougAlthough bacterial LPS is a well-defined inflammatory mediator, this molecule is also known as one of the best characterized monocytic mitogens, which, following interaction with its receptor CD14, induces first proinflammatory cytokines such as IL-1 and TNF-a and then anti-inflammatory cytokines such as IL-10. Regulation of pro- and anti-inflammatory cytokines has been suggested to be involved in distinct signaling pathways. For example, a recent study demonstrated IL-10 production in the LPS-activated THP-1 cells by transcriptional activation from the promoter sequence containing the consensus sequence for Sp1 and PD98059 failed to modulate IL-10 production . Not surCurrently, corticosteroids are the most common pharmacological drugs to control inflammation in the clinic. But, these drugs have significant side effects, especially for long-term use. Therefore, there are serious interests in developing more efficient and safer anti-inflammatory drugs. By using LPS as a stimulus, our results demonstrate that the URG extracts showed a higher biological activity than the RG extracts. This could be explained by the fact that the soluble hot-water extract of URG contains the higher concentrations of the ginsenosides than that of RG, indicating that URG may allow the easier and more efficient extraction of some known or other as-not-yet characterized active components in red ginseng than RG. In previous studies, it is known that the major ingredients of ginseng roots are the ginsenosides, which have various physiological activities including anti-inflammatory activity, anti-allergic, endothelium-independent aorta relaxation, and anti-tumor effects –24. In oIn conclusion, there seems a clear inhibitory effect of red ginseng on LPS-induced cytokine expression in cells because both URG and RG could inhibit expression of the major pro- and anti-inflammatory cytokines in the LPS-stimulated THP-1 cells. URG was more efficient than RG in this inhibitory effect because of the size differences: the average particle size of RG was about 127 μm whereas that of URG was about 3.5 um. Our demonstration of the higher inhibitory activity on LPS-induced cytokine expression of URG than RG suggests that the ginsenosides as well as other uncharacterized, biologically active components in red ginseng may be easily released out of the URG by standard hot-water extraction, compared to RG. Therefore, our HAC-gearshift system can be applied for other medicinal herbs because it provides an alternative and more efficient formulation of medicine.RG was further pulverized into an ultra-fine granule URG using the HAC Gear Shift System . This syURG and RG were prepared by decocting the dried herbs in boiling water (30 g/L) for 2 h as following: briefly, the extracts were freeze-dried for 48 h , dissolved in the phosphate buffered saline (PBS), filtered using a 0.45μm-filter unit , and stored at 4° C until used. Before the assay, the dried extract was dissolved in distilled deionized water and vortexed for 2 minutes at room temperature . These dURG and RG were embedded into pure embedding media in the beam capsules. The embedded blocks were kept at 50° C for 12–14 hours. The temperature was raised to 60° C and kept for 24–48 hours. The blocks were trimmed and thick sections (0.5 to 2.0 m) were cut to observe under an optical microscope. The area to be examined under TEM was selected and the blocks were further trimmed. The ultra thin sections (2 μm) were lifted onto a carbon grid and double-stained with uranyl acetate and lead acetate. The grids were placed in a watch glass containing a small amount of uranyl acetate and lead citrate. The grids were placed in a watch glass containing small amount of uranyl acetate and stained for 10–15 minutes. The grids were washed in two lots of 50% ethanol and two lots of double distilled water with continuous agitation. After drying on the filter paper, the grids were placed in a watch glass containing a few mL of lead citrate and stained for 5 to 10 minutes. The grids were washed briefly in 0.02 M sodium hydroxide and then in two lots of distilled water. After drying the grids, they were applied for TEM analysis.The soluble hot-water extracts of URG and RG were further analyzed at NT&BT (Korea) for their biochemical compositions by HPLC equipped with a C18 column and an auto-sampler Waters 717 plus.2.The THP-1 cells, known as the human monocytic leukemia cells, were obtained from Korean Cell Line Bank . The cells were prepared and resuspended in RPMI 1640 medium  supplemented with 10% (v/v) heat-inactivated fetal bovine serum  and 1% antibacterial antifungal solution (Gibco). They were maintained in a humidified incubator  at 37° C in presence of 5% CO160 nM PMA (Sigma) in RPMI medium was added to differentiate the THP-1 cells for 72 h at 37°C for all the cytokine assays through this study. The criteria for the differentiation of the THP-1 cells, adherence, morphological changes, and changes in expression of the cell surface makers such as integrin, FcγRI, CD4, and MHC class II antigen were applied. The PMA solution was prepared by dissolving PMA in sterile dimethylsulfoxide (DMSO) (Sigma). The stock solution was stored frozen at −20° C. Immediately prior to use, the PMA stock solution was diluted in RPMI medium to 160 nM at the final concentration.Cell viability was determined by using the 3--5(3-carboxymeth oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) kit  according to the manufacture's directions . For ana2 (1 × 106 cells) for experiments. The cells were pre-treated with 160 nM of PMA for 72 h and subsequently stimulated with 5 μg/mL of E. coli LPS with or without URG, RG, or both for 2 h in a humidified incubator  at 37° C in presence of 5% CO2.The THP-1 cells were seeded onto an φ 100mmTotal cellular RNAs were isolated from the THP-1 cells treated with Trizol (Gibco). The cDNA was synthesized from 1 μg of total RNA using 200 U Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen) with a total reaction volume of 20 μL. The resultant cDNA was amplified using the gene-specific primers . The ampAll the results were presented as the means ± SEM. One-way ANOVA with Tukey’s HSD test was used to determine their significance. Statistical analysis was carried out using the GraphPad Prism program (Prism 4.0 for Windows). The significance was defined with the following symbols: * P<0.05, ** P<0.01, *** P<0.001 against to LPS treatment sample and #<0.05, ##<0.01, ###<0.001 against to control.


Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens, so knowledge of how it competes against other LAB species is likely to prove invaluable.Many Gram-positive lactic acid bacteria (LAB) produce anti-bacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. These peptides fall structurally into three different classes, I, II, III, with class IIa being pediocin-like single entities and class IIb being two-peptide bacteriocins. Self-protective cognate immunity proteins are usually co-transcribed with these toxins. Several examples of cognates for IIa have already been solved structurally. S. pyogenes, (PDB:2fu2), and found it to comprise an anti-parallel four-helix bundle that is structurally similar to other bacteriocin immunity proteins. Sequence analyses indicate this protein to be a possible immunity protein protective against class IIa or IIb bacteriocins. However, given that S. pyogenes appears to lack any IIa pediocin-like proteins but does possess class IIb bacteriocins, we suggest this protein confers immunity to IIb-like peptides.We have solved the crystal structure of the gene-product of locus Spy_2152 from in silico characterization of a new putative immunity protein from S. pyogenes, possibly the first structure of an immunity protein protective against potential class IIb two-peptide bacteriocins. We have named the two pairs of putative bacteriocins found in S. pyogenes pyogenecin 1, 2, 3 and 4.Combined structural, genomic and proteomic analyses have allowed the identification and  Listeria and some have already been tested as food preservatives for controlling food-borne pathogens [Many Gram-positive bacteria produce anti-bacterial peptides and small proteins, called bacteriocins. There are three main classes produced by Gram-positive lactic acid bacteria (LAB): class I bacteriocins are the lantibiotics, small <4 kDa), post-translationally modified peptides containing unusual amino acids such as lanthionine; class II are small, unmodified, heat-stable bacteriocins <10 kDa); class III include larger >30 kDa) heat-labile proteins, such as murein hydrolases 0 kDa; cl. The gen0 kDa hea kDa, posathogens -7; they The class IIb two-peptide unmodified bacteriocins, for example plantaricin E/F , need thThe cyclic class IIc bacteriocins are characterized by being tryptophan-rich and lacking any GG, GxxxG or YGNGVxCxxxxC motifs, ; the claIn order to neutralize the toxic effect of the peptide on the "producing" cell the genes encoding bacteriocins are generally co-transcribed with a cognate immunity protein. These small proteins  interact very tightly with a specific bacteriocin or pair of bacteriocins and protect the "producing" microbe from the toxic effect of its own bacteriocin . The immThe sequencing of bacterial genomes, including those from human pathogens, has revealed a number of genes which might potentially code for new bacteriocins and immunity proteins, suggesting that the use of these antimicrobial peptides is more widespread than previously thought and that bacteria might be targeting several different bacterial species using these toxins. Understanding which specific immunity protein neutralizes which bacteriocin toxin is important if these peptides are to be used as antimicrobials.Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens. It causes a wide range of both minor diseases such as pharyngitis, erysipelas and pyodermas, that are readily controlled by antibiotics, as well as major, often lethal, conditions such as acute rheumatic fever, necrotizing fasciitis and streptococcal toxic shock syndrome, in developing countries and in the western world [The Gram-positive bacterium rn world . The seaS. salivarius producing a variety of bacteriocin-like inhibitory substances showing in vitro inhibitory activity against S. pyogenes, including Salivaricin A [S. pyogenes population in human microbiomes. Bacteriocin-like toxins and antitoxins may well have an impact on the development of new antibacterial strategies and treatments. A thorough understanding of the biology of the bacteriocins in combination with their immunity proteins is important for any possible therapeutic use of bacteriocins. The full sequences of LAB genomes provide opportunities to scan for the presence of toxins and their corresponding immunity proteins. However, the sequence alone may not be sufficient to identify these proteins.Other streptococcal species have been shown to secrete bacteriocin-like toxins, as there are reports of aricin A ,26. SuchS. pyogenes M1 GAS), named pyogenecin immunity protein Sp-PIP, determined at 2.15 Å resolution. We provide structural and sequence analyses, and identify the putative corresponding bacteriocin-like toxins in the S. pyogenes genome, which are found to belong to the class IIb two-peptide bacteriocins.Here we present the first structure of the protein from locus Spy_2152  method using selenomethionine (SeMet)-substituted protein. The model was refined to 2.15 Å with an R-factor of 15.7% and an R-free of 25.4%. The crystal asymmetric unit contains 78 (out of the 102) well ordered residues and 74 water molecules. The first methionine and the 23 C-terminal residues are disordered and cannot be identified in the electron density maps. The structure is high quality; all non-glycine and non-proline residues of the model lie either in the most favorable region (95.9% of residues) or in the additionally allowed region (4.1% of residues) of the Ramachandran plot. Detailed refinement statistics and crystallographic data are shown in Table We have expressed the pyogenecin immunity protein from nd (1tdp), 10th (2bl8) and 23rd (2k19) in the list of DALI matches, and the results are shown in Table 1tdp, over 75 residues, and, using the structure comparison service SSM at European Bioinformatics Institute http://www.ebi.ac.uk/msd-srv/ssm[Structural comparisons, using the DALI server , reveal p), 10th bl8 and 2d-srv/ssm, the RMShttp://pfam.sanger.ac.uk//family/Pf08951.Guided by the structure of Sp-PIP, whose sequence had not been assigned to any known protein family, we sought to expand our knowledge of its sequence relatives using the Pfam database . Having Enterococcus faecium (UniProt:Q3Y319) and closely related species carry a C-terminal PMSR domain - peptide methionine sulfoxide reductase (Pfam:PF01625) - as well as the N-terminal immunity protein domain.We investigated whether any other protein domains were to be found on any immunity proteins in the EntA_Immun family. Some members of the family do also carry associated domains. One protein contains an N-terminal helix-turn-helix transcriptional regulator (UniProt:Q88Y45), and a group of methionine sulfoxide reductases from S. pyogenes, including M1 GAS, MGAS10750, and MGAS8232, to try to identify any complementary bacteriocins and immunity proteins. We searched the proteomes with HMMs of both the known class I, antibacterial18 (Pfam:PF08130), and class II bacteriocins, bacteriocin_II (Pfam:PF01721), bacteriocin_IIc (Pfam:PF10439) as well as with the EntA_Immun families, from the Pfam database (release 23.0). We have identified a small genomic region containing four genes encoding putative bacteriocin proteins of class IIb , and have called the proteins Pyogenecin 1, 2, 3, and 4, respectively . The organization of this region is shown in Figure S. pyogenes genome: Spy_2152 (Sp-PIP) and Spy_1675. Both encoded proteins were found in all the S. pyogenes strains examined, and are identified as belonging to the EntA_Immun family (Pfam:PF08951), a family which contains nearly 200 different members from LAB and closely related species.Sp-PIP belongs to a large family of putative bacteriocin immunity proteins. To investigate the possible targets of the presumed immunity protein we carried out an analysis of the proteomes of several strains of The four-helix bundle structure of Sp-PIP was found to be most closely related to those of a number of pediocin-like IIa immunity proteins. Three subgroups of pediocin-like IIa immunity proteins have been defined on the basis of common sequence motifs and phylogenetic analysis , being d2bl8• Group A: EntA-im, the enterocin A immunity protein, ; PDB ID:2k19• Group B: PisI, the piscicolin 126 immunity protein ; PDB ID:1tdp• Group C: ImB2, the carnobacteriocin B2 immunity protein ; PDB ID:The structural comparison results suggest that Sp-PIP is most closely related to Imb2, from subgroup C. These results suggest that Sp-PIP is highly likely to function as a bacteriocin immunity protein with similarities to proteins from group C.S. pyogenes genome identified another gene locus encoding a protein matching the EntA_Immun family. Both of these loci were seen in all strains examined. Unlike the case in many other species, in S. pyogenes these bacteriocin immunity proteins were not found closely linked to their bacteriocins, a condition that might be explained by the remnants of a transposon identified between them.Sequence analysis reveals that Sp-PIP belongs to the now expanded Pfam family of immunity proteins EntA_Immun that also includes pediocin-like immunity proteins from groups A, B and C as detailed above, thus confirming its evolutionary link to known bacteriocin immunity proteins. A search for other immunity proteins of a similar nature in the Functions for proteins may often be inferred through consideration of the combinations of the functional domains found on them. A number of domain-pairings were identified on some of the immunity family members, such as an N-terminal helix-turn-helix transcriptional regulator on one protein, which might be suggestive of immunity proteins acting as transcriptional regulators or of interacting with transcription regulators and modulating their function. A group of methionine sulfoxide reductases are found to carry both a C-terminal PMSR domain and the immunity domain. The PMSR domain is involved in reversing protein inactivation by oxidation of methionine residues. However, it is not clear what, if any, functional significance these domain-pairings might have.S. pyogenes contains a class I lantibiotic system composed of one unique lantibiotic type A sequence called srtA and just one unique immunity protein srtI. These two genes are closely associated on the genome, but are separated from the proteins of the class II system described below as well as from the presumed immunity proteins. This suggests that the Sp-PIP is unlikely to act as an immunity protein for class I bacteriocins. Class II bacteriocins are subdivided into classes IIa, IIb, IIc and IId. We did not find any bacteriocins of class IIa encoded by the S. pyogenes genome, which would have the well-conserved N-terminal YGNGVxCxxxC motif [The search for potential bacteriocin targets for this putative immunity protein took in classes I, II and III. Our analyses identified that xC motif ,31. SearxC motif  and circxC motif  do not sxC motif . As withBased on the sequence similarity to other two-peptide bacteriocins, the genomic arrangement of the genes found, and the identification of two immunity proteins, we hypothesize that the putative pyogenecins 1 and 2 associate to form one active bacteriocin and pyogenecins 3 and 4 associate to form a second active bacteriocin possibly targeting a different bacterial species. These peptides need now to be synthesized to allow testing of their biological activity in a suitable bacteriocin-assay. Given that we find two immunity proteins we suggest that each one is specific for one of the putative bacteriocin pairs, Pyogenecin1/Pyogenecin2 and Pyogenecin3/Pyogenecin4.There is also the possibility that one or both of these immunity proteins might be an orphan immunity protein , so-callin silico characterization of a new putative immunity protein from S. pyogenes. Structurally similar proteins seem to provide immunity protection for single peptide IIa class bacteriocins. Further biological and biochemical studies are needed to verify the antibacterial activity of the putative pyogenecins and to determine the degree of resistance and cross-resistance provided by the proposed immunity protein.The structure of the putative immunity protein Sp-PIP protective against potential class IIb two-peptide bacteriocins could be the first structural representative of this class. A combined structural, genomic and proteomic analyses has allowed the identification and S. pyogenes M1 DNA with KOD DNA polymerase using conditions and reagents provided by the vendor . The gene was cloned into the pMCSG7 vector using a modified ligation-independent cloning protocol [E. coli BL21 (DE3) - Gold (Stratagene) harboring an extra plasmid encoding three rare tRNAs . The pMCSG7 vector bearing a tobacco etch virus (TEV) protease cleavage site creates a construct with a cleavable His6-tag fused onto the N-terminus of the target protein and adds three artificial residues (Ser-Asn-Ala) on that end. The cells were grown using SeMet-containing enriched M9 medium and conditions known to inhibit methionine biosynthesis. The cells were grown at 37°C to an OD600 of ~0.6 and protein expression induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG). After induction, the cells were grown overnight with shaking at 20°C. The harvested cells were re-suspended in lysis buffer buffer  in the presence of lysozyme (1 mg/mL) and of protease inhibitor cocktail (100 μL per 2 g of wet cells), and then kept on ice for 20 min before sonication. Then the cells were lysed using sonication. The lysate was clarified by centrifugation at 30,000 × g  for 20 min, followed by filtration through 0.45 μm and 0.22 μm in-line filters (Gelman). The lysate was then applied to a 5 mL HiTrap Ni-NTA column on the AKTAxpress . His6-tagged protein was eluted using buffer containing a higher concentration of imidazole , and the His6-tag was cleaved from the protein by treatment with recombinant His6-tagged TEV protease. A second Ni-NTA affinity chromatography was performed manually to remove the His6-tag and His6-tagged TEV protease. A total of 90 mg of protein was purified for finding crystallization conditions. Then the protein was dialyzed in 20 mM Tris-HCl pH 7.1, 50 mM NaCl, 2 mM dithiothreitol (DTT) and concentrated to 110 mg/mL using a Centricon Plus-20 Centrifugal Concentrator (Millipore). The standard purification protocol has been thoroughly described previously [The ORF from the Spy_2152 locus was amplified from genomic protocol  and overeviously .The initial crystallization conditions were searched using the sitting drops vapor diffusion method at 18°C with the help of the Mosquito crystallization workstation (TTP Labtech) with Index (Hampton Research) and Wizard I and II  crystallization screens. Crystals suitable for X-ray experiment were obtained at the initial stage of crystal screening, the crystallization condition number 23 of Wizard II within several days. Crystals were flash-frozen in liquid nitrogen with crystallization solution containing 25% (v/v) glycerol as cryoprotectant prior to data collection.a = 76.09 Å, b = 30.28 Å, c = 35.86 Å, α = 90.00°, β = 113.26°, and γ = 90.00°. The diffraction data were processed using the HKL3000 suite of programs [Anomalous diffraction data were collected at the selenium peak from crystals of a SeMet-substituted protein. The data sets were collected on an ADSC quantum Q315 charge-coupled device-detector at 100 K on the Structural Biology Center beamline 19ID at the Advanced Photon Source, Argonne National Laboratory. The space group was found to be C2, with cell parameters of programs .2fu2.All procedures for single wavelength anomalous dispersion (SAD), phasing, phase improvement by density modification, and initial protein model building were done using the structure module of the HKL3000 software package. The mean figure of merit of the phase-set was 0.207 for 50-2.25 Å data and improved to 0.542 after density modification (DM). The autotracing Arp/wArp module  in HKL30CC, LV, MC performed the crystallography part of the research; AB, PC and RF performed the genome sequence searches; PC, AB, ZO, WM and AJ analyzed data; AJ designed the research; PC, AB and AJ wrote the manuscript. All authors read and approved the final manuscript.


HLA-DRB1 alleles (N = 18), 2) RA cases who have two high-risk HLA-DRB1 alleles (N = 163), and 3) controls who have two low-risk HLA-DRB1 alleles (N = 652). We hypothesized that since Group 1's RA was likely due to non-HLA related risk factors, and because Group 3, by definition, is unaffected, comparing Group 1 with Group 2 and Group 1 with Group 3 would result in the identification of candidate susceptibility loci located outside of the MHC region. Accordingly, we restricted our search to the 21 non-chromosome 6 autosomes. The case-case comparison of Groups 1 and 2 resulted in the identification of 17 SNPs with allele frequencies that differed at p < 0.0001. The case-control comparison of Groups 1 and 3 identified 23 SNPs that differed in allele frequency at p < 0.0001. Eight of these SNPs  were common to both lists.We conducted a search for non-chromosome 6 genes that may increase risk for rheumatoid arthritis (RA). Our approach was to retrospectively ascertain three "extreme" subsamples from the North American Rheumatoid Arthritis Consortium. The three subsamples are: 1) RA cases who have two low-risk  Rheumatoid arthritis (RA) is a chronic inflammatory disorder in which the articular joints are gradually destroyed. Occasionally there is systemic involvement, which can include pulmonary fibrosis and vasculitis in various organs. The etiology of RA is complex, with significant genetic and environmental components.HLA-DRB1 locus as the leading suspect [DRB1*0401, *0404, *0405, *0408, and *0409), while other alleles appear to confer a moderate increase in risk . In what follows we denote the high-risk alleles as H, the moderate risk alleles as M and the low-risk alleles as L. Most of the high-risk alleles possess a shared epitope (SE) of five amino acids at positions 70-74 in the third hypervariable region of the DRβ1chain [*0401/DRX  and *0404/DRX are estimated to increase the relative risk of RA by 4.7-fold and 5.0-fold, respectively, while the *0401/*0401 genotype carries a relative risk of 18.8 and the compound heterozygote *0401/*0404 has a relative risk of 31.0 [4/4 or 4/*0401.Among the genetic components, genes in the MHC region on chromosome 6p21.3 are widely acknowledged to be the major player, with the DRB1 alleles is much smaller than 30%. We hypothesize that RA cases with two non-SE encoding DRB1 alleles constitute a subgroup of patients that are enriched for other susceptibility alleles located elsewhere in the genome. Accordingly, we undertook a genome-wide association (GWA) analysis that compares single-nucleotide polymorphism (SNP) allele frequencies in two groups of cases-those whose DRB1 genotype contains two high-risk alleles (HH) and those whose DRB1 genotype contains two low-risk alleles (LL)-and one group of controls whose DRB1 genotype also consists of two LL alleles.Not all persons with RA possess one or two high-risk SE alleles, however. It is estimated that in persons of European ancestry, approximately 30% do not carry an SE-encoding allele . In the HLA-DRB1 genotypes in cases and controls from the North American Rheumatoid Arthritis Consortium (NARAC). There were an additional 69 cases and a single control for whom DRB1 genotypes were not available. Table 2 = 772.2, p = 1.2 × 10-164). The table also shows that in the NARAC sample there are very few cases with the low-risk LL genotype (2.25%). Because of the very small LL case sample, we chose to use Fisher's exact test for all comparisons, realizing the statistic is severely conservative when interpreted with reference to conventional alpha levels [HLA-DRB1 genotypes, we restrict our attention to the 21 non-chromosome 6 autosomes.Table th SNP from all of the autosomes, except chromosome 6, for a sample of 9,920 SNPs, and performed an EIGENSTRAT analysis [There is some evidence for population substructure in this sample-perhaps occasioned by the fact that patients were drawn from rheumatology clinics across North America, while all of the controls were selected from participants who were part of the New York Cancer Project . We searanalysis .p = 0.01). Despite the unequal variances, no differences were seen in the mean values for the two continuous clinical variables anti-cyclic citrullinated peptide (anti-CCP) and rheumatoid factor IgM titers. Unfortunately, no data were made available for an important behavioral/environmental variable, namely smoking.Table p < 0.0001. The comparison of the LL cases and the LL controls resulted in the identification of 23 SNPs. Eight SNPs are common to both lists. Also reported in Table Table Figure N = 18 cases is extremely small by today's standards. Nonetheless, we are mindful that even small samples can be useful if the genetic effects are large enough. For instance, Cudworth and Woodrow [We are, of course, aware that a sample of  Woodrow  were ablAlthough it is customary to report SNP allele frequencies in terms of the MAF  or are close to the same gene . We carried out an analysis of linkage disequilibrium and estimated the r2 values between these adjacent SNPs from the NARAC control panel and obtained estimates of r2 = 1.0 for the two SNPs near FBXL17, r2 = 0.384, 0.949, 0.821, 0.801, and 0.734 for the five pairs of adjacent SNPs from TRIO (from rs173948 to rs27114), and r2 = 0.975, 0.837, 0.997, and 0.960 for the four pairs of adjacent SNPs near SLC6A15.Some of the SNPs listed in Table Table p < 0.0001, respectively. Eight SNPs are common to both lists, although the presence of significant linkage disequilibrium suggests that the actual overlap would be less. We used a variety of sources to identify the nearest gene, or predicted gene [We carried out a GWA study using an extreme sampling design on three subsets of the NARAC data. We identified 17 SNPs and 23 SNPs that distinguished the LL cases from the HH cases, and the LL cases from the LL controls at ted gene -19. For GWA: genome wide association; H: High-risk allele; L: Low-risk allele; M: Moderate-risk allele; MAF: Minor allele frequency; NARAC: North American Rheumatoid Arthritis Consortium; RA: Rheumatoid arthritis; SE: Shared epitope; SNP: Single-nucleotide polymorphism.The authors declare that they have no competing interests.BKS conceived and designed the study and drafted the manuscript. All of the authors participated equally in managing the data and in the statistical analysis of the data.